Method of prognosticating respiratory bacterial infection using urokinse receptor measurement

ABSTRACT

Method of diagnosing and/or prognosticating HIV infection in a subject comprising the steps of: (a) performing in vitro a measurement of the level of a marker in the form of (i) urokinase plasminogen activator receptor (uPAR), (ii) soluble urokinase plasminogen activator receptor (suPAR), (iii) urokinase-type plasminogen activator (uPA), (iv) one or more degradation products of (i), (ii) or (iii), and/or (v) an mRNA for (i), (ii) or (iii), in a biological fluid sample from a subject, and (b) using the measurement value obtained to evaluate the state of the subject.

TECHNICAL FIELD OF THE INVENTION

The present invention concerns the diagnosis and/or prognosis of majorrespiratory bacterial pathogens. In particular it concerns Streptococcuspneumoniae and Mycobacterium tuberculosis. More particular it concernsthe measurements of the concentration of soluble urokinase plasminogenactivator receptor (suPAR) in human biological fluids (sputum, cysticfluid, ascites, serum, plasma, urine) as a tool of diagnosingrespiratory bacterial infection as well as the prognosis of diseaseprogression.

BACKGROUND OF THE INVENTION

The cellular receptor for urokinase (uPAR, CD87) plays multiplefunctions in cell migration, cell adhesion, pericellular proteolysis andtissue remodeling. uPAR is expressed by most leukocytes includingmonocytes, macrophages, neutrophils and platelets. uPAR is an activationantigen in monocytes and T cells and T-cells from HIV-1 infectedindividuals express elevated levels of uPAR¹, 1994. HIV-1 infection ofleukocytes in vitro causes up-regulation of uPAR cell surface expressionin a process which appear to be coordinated temporally with the onset ofviral replication².

uPAR may be shed from the cell surface generating a soluble form of thereceptor (suPAR) lacking the GPI-anchor. The shedding mechanism ispoorly understood but may occur by GPI-specific phospholipase Dcatalyzed hydrolytic cleavage of the GPI-anchor). Soluble forms of uPAR(suPAR) has been identified in cell culture supernatants and in diversebiological fluids such as tumor ascites, cystic fluid, serum, plasma andrecently also in urine ³.

Serum, plasma and urine levels of suPAR are elevated in patientssuffering from different types of cancer⁴, the paroxysmal nocturnalhemoglobinuria syndrome (PNH) syndrome⁵, and in rheumatoid arthritispatients⁶. The plasma level of suPAR is furthermore a prognostic markerfor overall survival in patients suffering from HIV-1 infection⁷.

The cellular origin of circulating suPAR is not known. Many, if not all,cells which express uPAR also shed soluble forms of the receptor whencultured in vitro. The source of excess serum suPAR in cancer patientshas been suggested to derive from the cancer cells and/ortumor-infiltrating macrophages as these cells often express high levelsof uPAR and experiments using xenografted mice carrying human tumorshave indeed demonstrated that the tumor tissue does release suPAR to thecirculation and urine⁸.

SUMMARY OF THE INVENTION

The technical problem addressed by the present invention is to provide anovel marker for diagnosing and prognosticating major respiratorybacterial pathogens, in particular Streptococcus pneumoniae andMycobacterium tuberculosis. A further technical problem addressed by thepresent invention is to provide a marker of the said type, which issimple and affordable to measure.

The present invention has provided a solution to the above technicalproblems, the invention being directed to a method of diagnosing orprognosticating major respiratory bacterial pathogens, such asStreptococcus pneumoniae and Mycobacterium tuberculosis in a subjectcomprising the steps of

(a) performing in vitro a measurement of the level of one or moremarkers in the form of (i) urokinase plasminogen activator receptor(uPAR), (ii) soluble urokinase plasminogen activator receptor (suPAR),and/or (iii) one or more degradation products of (i) or (ii), in abiological fluid sample from a subject, and

(b) using the measurement value obtained to evaluate the state of thesubject.

The invention is based on the surprising discovery that soluble uPAR(suPAR) is present in elevated levels in serum, plasma and urine ofpatients with two major respiratory bacterial pathogens, Streptococcuspneumoniae and Mycobacterium tuberculosis, and that the level of suPARis useful as a diagnostic marker. Also, the level of suPAR inindividuals with two major respiratory bacterial pathogens,Streptococcus pneumoniae and Mycobacterium tuberculosis is prognosticfor the development of the disease and death. The suPAR level is a noveland highly diagnostic and prognostic factor, even in the context ofother known prognostic factors related to Streptococcus pneumoniae orMycobacterium tuberculosis infection of the lung.

Furthermore, the present invention is based on the recognition that theamount of suPAR is correlated to the amount of uPAR and that thereforethe amounts of uPAR is equally suitable as diagnostic and prognosticindicators of tuberculosis infection.

A further advantage of the invention is that measurement of suPAR can beperformed using e.g. a simple ELISA technique or even a stick and maytherefore provide a very inexpensive, simple and quick supplement to thecurrently used prognostic tools for persons infected with tuberculosis.Thus, in developing countries without the financial possibility to carryout the costly assays used in the western world, suPAR levels could beused 1) to determine tuberculosis status (diagnosis), 2) to selectpatients for treatment (prognosis), and 3) to monitor the progress oftreatment.

Furthermore, the present invention involves the advantage that whilesuPAR levels can be measured very simply, using e.g. a urine sample orsputum sample, which is easy to obtain, conventional diagnostic andprognostic tests for e.g. tuberculosis are uncertain, complex andinvolve e.g. growth in culture of sputum, clinical testing and chestx-ray.

The invention further relates to a method of evaluating the progressionof the state of a subject suffering from a major respiratory bacterialpathogen, such as tuberculosis or Streptococcus pneumoniae comprisingthe steps of

(a) performing in vitro a measurement of the level of one or moremarkers in the form of (i) urokinase plasminogen activator receptor(uPAR), (ii) soluble urokinase plasminogen activator receptor (suPAR),and/or (iii) one or more degradation products of (i) or (ii), in each ofa number of biological fluid samples from a subject, wherein the samplesare obtained at different points in time, and

(b) using the measurement values obtained to evaluate the progression ofthe state of the subject.

This method may be used to continuously monitor the state e.g. thetreatment efficacy of the patient.

Finally, the present invention relates to an ELISA-kit and stick-kitutilizing the above knowledge.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. suPAR levels among TB sputum positive (SP), TB culture positive(CP), presumed TB (PTB), TB positive receiving treatment at enrolment(TAE), and TB negative (NEG). The box-plot shows the median (black linein box), quartiles (box) and outliers (cases with values between 1.5 and3 box lengths from the upper or lower edge of the box), but not extremes(cases with values more than 3 box lengths from the upper or lower edgeof the box).

FIG. 2 a shows the relationship between suPAR values and the probabilityof being diagnosed TB positive. The 16 patients who were in treatmentfor TB at time of inclusion were not included in the analysis. Thus,included in the analysis are the TB positive (TB sputum and culturepositive and presumed TB) and the TB negative individuals. Patients withincreasing suPAR have higher probability of being diagnosed with activeTB (P=0.0001). Patients with suPAR values above 8.3 ng/ml (n=8) are notshown. The blue lines at the bottom indicate the individual patientsuPAR values and the high line is the median value. The stippled linesare 95% confidence intervals.

FIG. 2 b. Probability of being diagnosed of TB when excluding the TBsputum positive. Patients included in the probability analysis are the35 culture positive, the 63 presumed TB and the 64 TB negative. Thus, itpredicts the probability of all TB suspects excluding the microscopicpositive and the 16 patients in treatment at enrollment. Eight sampleshad suPAR values above 8.5 ng/ml and are not shown. The figure showsthat the probability of being TB positive despite a TB negative sputummicroscopic analysis increases with increased suPAR levels (p=0.001).The blue lines at the bottom indicate the individual patient suPARvalues and the high line is the median value. The stippled lines are 95%confidence intervals.

FIG. 2 c. The figure is identical to FIG. 2 b, except for the exclusionof HIV-1 infected individuals. There was a significant relationshipbetween increasing suPAR and probability of being diagnosed as culturepositive or presumed TB (p=0.02). The blue lines at the bottom indicatethe individual patient suPAR values and the high line is the medianvalue. The stippled lines are 95% confidence intervals.

FIG. 2 d shows the probability of being TB positive among HIV-1 positiveaccording to suPAR value. p=0.08. There was a trend towards arelationship between increasing suPAR and probability of being diagnosedas culture positive or presumed TB (p=0.08). The blue lines at thebottom indicate the individual patient suPAR values and the high line isthe median value. The stippled lines are 95% confidence intervals.

FIG. 3. Kaplan Meier survival curves showing survival among the 182patients with active TB. The thin line indicates patients with low suPARand bold line patients with high suPAR. Figure A: There was nosignificant difference when stratifying patients according to medianvalue. Figure B: Patients with suPAR values above 2 times the medianvalue (i.e., >4.2 ng/ml, n=38), died significantly faster that patientswith suPAR below 2 times the median value (n=144), p=0.007, log ranktest.

FIG. 4. After treatment, sera were available from 101 individuals. Thebox-plot shows the pre- and post (post marked with −P) suPAR levels ofthese 101 individuals. A: TB sputum positive (SP), B: TB culturepositive (CP), C: presumed TB (PTB), D: TB positive receiving treatmentat enrolment (TAE), and E: TB negative (NEG). For simplicity, thebox-plot shows the median (black line in box) and quartiles (box), butnot outliers and extremes (cases with values more than 1.5 box lengthsfrom the upper or lower edge of the box).

FIG. 5. Box plots based on the median, quartiles, and extreme values ofplasma suPAR levels. A: Control group (median =2.6 ng/ml) andpneumococcal bacteremic patients (median =5.5 ng/ml). suPAR wassignificantly elevated in patients compared to controls (p=0.001). B:Patients surviving the infection (median =5.0 ng/ml) and patients dyingfrom the infection (median =9.4 ng/ml). The increased level of suPAR inthe group of dead patients was statistically significant (p=0.0001). Theboxes represent the interquartile range that contains the 50% of valuesand whiskers extending from the boxes the highest and lowest values,excluding outliers and extremes. Circles (o) represent outliers (1.5 and3 box-lengths) and stars (*) represent extremes (values more than 3 boxlengths).

FIG. 6. suPAR levels in individuals with suspected TB according towhether the patients were positive or negative for AFB in directmicroscopy.

FIG. 7. suPAR levels in untreated and treated TB patients.

FIG. 8. The Y-axis on the left shows the serum level of suPAR (ng/ml).The Y-axis on the right shows the percentage of cells expressing theuPAR receptor (CD87 positive cells).

DETAILED DESCRIPTION OF THE INVENTION

In the following, the invention is explained in particular withreference to suPAR for reasons of simplicity. This should not beunderstood as a limitation of the scope of the present invention tosuPAR. Furthermore, suitable extrapolations to uPAR and the degradationproducts of the said two substances lie well within the skill of aperson skilled in the art.

The inventors have surprisingly found that the concentration of suPAR, amolecule that is in general known to be involved in cell migration andadhesion, is increased in serum and/or sputum from two major respiratorybacterial pathogens, Streptococcus pneumoniae and Mycobacteriumtuberculosis persons compared with healthy controls.

Major respiratory bacterial pathogens may be selected from

-   Streptococcus pneumoniae (including drug-resistant S. pneumoniae)-   Chlamydia pneumoniae and Chlamydia psittaci-   Legionella spp. Coxiella burnetii-   Haemophilus influenzae Respiratory viruses-   Enteric Gram-negative bacilli (specially Klebsiella spp.)-   Endemic fungi (coccidioidomycosis, histoplasmosis, blastomycosis)b-   Staphylococcus aureus-   Streptococcus pyogenes-   Mycoplasma pneumoniae-   Mycobacterium tuberculosis-   Pseudomonas aeruginosaa-   Pneumocystis cariniib.

The biological fluid sample may be any fluid that can be obtained fromhumans i.e. sputum, cystic fluid, ascites, blood, serum, plasma, andurine. Urine is preferred due to the fact that it is easy to obtain.When urine is used as the biological fluid sample, the measurement ofthe marker should be correlated to the total concentration level of thesample, e.g. by correlating the measurement of the marker to the contentof creatinine in the sample.

Preferably, the biological fluid sample is stored at a temperature ofbelow 0° C., more preferably from −20° C. to −80° C., until measurement.

The measurement of the marker in the biological fluid sample may becarried out using any available method/device therefore. Examples ofsuch measurement methods/devices are ELISA, RIA (radioimmunoassay),western blotting, TR-FIA (Time-Resolved ImmunoFluorometric Assays), FACSanalysis, sticks, etc. Preferred measurement methods/devices are ELISAand sticks.

An ELISA may be carried out in a number of different embodiments, manyof which are applicable in the present invention. One ELISA embodiment,which is particularly suitable for use in the present invention, is theone described by⁸, and⁹ which are included herein by this reference.

The measurement of the marker may be carried out using any suitablestick. Preferably, the stick is a stick comprising an antibody to themarker as a capture agent.

Preferably, the measurement of uPAR in the biological fluid sample iscarried out by FACS analysis, western blotting or ELISA.

Preferably, the measurement of uPAR/suPAR degradation products iscarried out using western blotting or TR-FIA (Time-ResolvedImmunoFluorometric Assays).

The measurement of uPAR is carried out in biological fluid samplescontaining uPAR expressing cells, i.e. blood samples.

The measurement value of the level of marker obtained in step (a) may beused to evaluate the state of the subject by comparing the measurementvalue with the level of the marker in subjects not having a bacteriallung infection with Streptococcus pneumoniae or Mycobacteriumtuberculosis.

As mentioned above, one aspect of the present invention relates to amethod of evaluating the progression of the state of a subject sufferingfrom Streptococcus pneumoniae or Mycobacterium tuberculosis infection.

The measurement values of the level of marker obtained in step (a) maybe used to evaluate the progression of the state of the subject bycomparing the measurement values with the level of the marker insubjects not infected with tuberculosis or Streptococcus pneumoniaeand/or by comparing the temporal course of measurement values with thatof subjects not infected with tuberculosis or Streptococcus pneumoniae.

The invention further relates to an ELISA-kit for evaluating thephysical state of a subject suffering from tuberculosis infectioncomprising a) an immobilised capture agent capable of capturing one ormore markers in the form of (i) urokinase plasminogen activator receptor(uPAR), (ii) soluble urokinase plasminogen activator receptor (suPAR),and/or (iii) one or more degradation products of (i) or (ii), and b) abinding partner capable of binding to the marker, the binding partnercomprising c) a label system.

The capture agent may be an antibody to the marker.

The binding partner may be an antibody to the marker.

Preferably, the label system is conjugated to the binding partner. Thelabel system may be any conventionally used label system, such asantibody to the binding agent conjugated to an enzyme, e.g. animmunoglobulin-alkaline phosphatase conjugate.

Furthermore, the invention relates to a stick for evaluating thephysical state of a subject suffering from tuberculosis or Streptococcuspneumoniae infection comprising a) an immobilised capture agent capableof capturing one/or more markers in the form of (i) urokinaseplasminogen activator receptor (uPAR), (ii) soluble urokinaseplasminogen activator receptor (suPAR), and/or (iii) one or moredegradation products of (i) or (ii) and b) a binding partner capable ofbinding to the said marker, the binding partner comprising c) a labelsystem.

The capture agent may be an antibody to the marker.

The binding partner may be an antibody to the marker.

Preferably, the label system is conjugated to the binding partner. Thelabel system may be any conventionally used label system, such asantibody to the binding agent conjugated to an enzyme, e.g. animmunoglobulin-alkaline phosphatase conjugate.

Definitions

In connection with the present invention “uPAR” is defined as any formof uPAR (same as CD87) present on the surface of uPAR expressing cellsin biological fluids.

The expression “uPAR expressing cells” refers to all cells expressinguPAR (CD87) such as monocytes, leucocytes, macrophages, andneutrophiles.

In connection with the present invention “suPAR” (soluble uPAR) isdefined as any form of uPAR (same as CD87) present in biological fluidsin a non-cell-bound form.

“Biological fluids” are defined as any fluid that can be obtained fromhumans i.e. sputum, cystic fluid, ascites, blood, serum, plasma, andurine.

The expression “degradation product of suPAR or uPAR as used hereinmeans all fragments thereof observed using western blotting orantibodies against suPAR or uPAR. In particular, the said expressionincludes the D1, D2 and D3 fragments of suPAR.

EXAMPLES Example 1 Diagnosis of Active TB by Measurement of Serum suPAR

Aim:

To determine whether patients suspected of having active TB has elevatedlevels of suPAR in serum, i.e. whether suPAR levels may be used todiagnose active TB.

Description of the Experiment

Mycobacterium Tuberculosis is an intracellular pathogen that residespredominantly within macrophages. The urokinase plasminogen activatorreceptor (uPAR) is mainly located on monocytes and macrophages. uPAR isa three domain glycosylated protein (D1-D3) which binds urokinase withhigh affinity through its D1 domain. The uPAR/uPA complex is involved inplasminogen activation, pericellular proteolysis and tissue remodellingas well as integrin activation, cell adhesion and migration. The uPAR isbound to the cell surface by a GPI anchor, which may be cleaved,resulting in a soluble form of the receptor (suPAR). Soluble forms ofuPAR (suPAR) include the full-length receptor (D1-D3) as well as a D2D3fragment with chemotactic properties¹⁰ and a D1 fragment with unknownfunction. suPAR has been identified in cell culture supernatants and inbiological fluids such as cystic fluid, serum, plasma and urine¹¹.

Due to the difficulties of diagnosing tuberculosis infection indeveloping countries and the high mortality caused by the infection, wehave investigated suPAR levels among individuals suspected of havingactive tuberculosis in a cohort in Guinea-Bissau. Here we show thatsuPAR carries both diagnostic and prognostic information in patientssuffering from active tuberculosis.

Population and TB Surveillance

Four suburban areas in Bissau, the capital of Guinea-Bissau, with around46,000 inhabitants are followed through a demographic surveillancesystem since 1978. In May 1996, a TB surveillance system was implementedin the area in co-operation with the national TB Hospital, HospitalRaoul Follereau (HRF). All adults permanently or temporarily living inthe study area, presenting at any of the 2 health centres with symptomsor signs of active tuberculosis were referred to the hospital forfurther investigation. Two TB nurses performed visits every third monthin houses where TB cases had been found, examining and interviewing thehousehold members in order to find secondary cases. Suspected cases werereferred to the TB-hospital for further medical examinations. Patientsliving in the area, but with TB-treatment initiated elsewhere, were alsoreferred for further investigation and inclusion if identified by thenurses distributing medicine within the national TB programme.

Inclusion Criteria

Criteria for inclusion in the study was one or more of the followingsymptoms and signs without other explanatory disease: cough >1 monthwithout improvement on antibiotics, fever constantly or periodically formore than 1 month, weight loss, dyspnoea, haemoptysis, nightly sweats orlymphadenopathy. Persons aged <15 years and pregnant women were notincluded. Consenting patients were investigated clinically, interviewedusing standardised questionnaires concerning medical history,socio-demographic and behavioural risk factors, investigated with chestx-ray, direct microscopy on morning sputum on 3 consecutive days, sputumculture and Mantoux-test (using Multiteste®, Bio-Merieux, France). Bloodwas drawn for testing of HIV-infection, haematology and immunologicalparameters. Two hundred and sixty-two patients with suspected TB wereincluded in this study. The patients were enrolled between 1996 and1998. 147 were men and 115 women. Mean age was 41.4 years (range 15-80).Based on the examinations, patients were divided in 4 groups: 1)Patients positive for AFB in sputum direct microscopy (Acid FastBacilli, AFB, in direct microscopy) were denoted TB sputum positive. 2)Patients negative in microcopy but positive in culture were denoted TBculture positive. 3) Patients with clinical signs, symptoms and x-raychanges compatible with active intrathoracic TB, but withoutbacteriological confirmation in sputum direct microscopy or culture,were treated with antibiotics (co-trimoxazole or amoxicillin) and thenre-evaluated clinically and with chest x-ray. If there was noimprovement and suspicion remained, the patient was assumed to have TBand denoted presumed TB. 4) Patients not diagnosed as having TB weretreated according to diagnose (e.g. pneumonia). These patients aredenoted TB negative.

TB Treatment

A 4-month intensive phase of daily Directly Observed Treatment (DOT)with standard doses of Ethambutol, Isoniazid, Rifampicin, andPyrazinamide was followed by a 4-month continuation phase withEthambutol and Isoniazid collected by the patient at the health centretwice per month. This treatment regimen was recommended for HIV infectedindividuals by the national TB programme in Guinea-Bissau when theresearch project was initiated in 1996; for reasons of confidentialityand comparability HIV-positive and HIV-negative individuals received thesame treatment. In addition, all patients were given Vitamin B complexand Multivitamins daily. Patients who presented with severe disease wereoffered hospitalisation if beds were available. In the intensive phase,patients failing to show up for treatment were visited the same day by anurse and encouraged to continue treatment. Adherence to treatment wasverified by pill count and an INH urine test at 2, 5 and 8 months offollow-up; information was noted on treatment cards and forms.

Laboratory Methods

At inclusion, morning sputum samples were collected during threeconsecutive days by a field assistant and transported in a sealedcontainer at 4° C. the same day to the National Public Health Laboratory(LNSP) in Bissau for direct microscopy and culture. Before culture,samples were digested and decontaminated from non-mycobacterialmicroorganisms with the lauryl sulphate method. A 0.5-mL aliquot of thehomogenised specimen was inoculated into one tube with conventionalLöwenstein-Jensen egg medium (LJ) and into one containing a modified LJwith 0.6% pyruvate. The tubes were incubated at 37° C. and examinedweekly for 7 weeks. Growth of mycobacteria was confirmed by acid-fastmicroscopy. Isolates were transported frozen to the Swedish Institutefor Infectious Disease Control (SMI) in Stockholm for confirmation.

HIV Testing

Sera were screened for HIV at LNSP using Capillus® HIV-1/HIV-2(Cambridge Diagnostics, Galway, Ireland) and Enzygnost® Anti-HIV 1+2Plus (Behring Diagnostics Gmbh, Marburg, Germany). Positive samples werethen confirmed with Multispot® HIV-1/HIV-2 (Sanofi Diagnostics Pasteur,Marnes-la Coquette, France). Dual reactive samples were sent frozen toSMI in Stockholm and confirmed using Immunocomb® II Hiv-1&2 Bispot(Orgenics, Yavne, Israel). CD4- and CD8-cell counts were measured usingthe Immuno-Alkaline phosphatase method¹³.

suPAR Measurement

Measurements of suPAR were performed retrospectively on frozen serumsamples using a sandwich ELISA. Immunoplates (Maxisorb, Nunc, Denmark)were incubated overnight at 4° C. with a murine monoclonal antibodyagainst human suPAR, R2 (2 μg/mL), in coating buffer (15.1 mM Na₂CO₃,35.7 mM NaHCO₃, pH 9.6). After incubating, the wells were washed threetimes with washing buffer (PBS, 0.1% Tween 20). Non-specific binding wasblocked with 2% bovine serum albumin (filtered BSA, Sigma Chemical Co.,St Louis, Mo., USA) in phosphate-buffered saline (PBS) for 30 minutes at37° C. (shaking). After washing three times, the plates were incubatedwith 10 ul of serum samples diluted with 90 μl buffer, pH 7.4 (7.3 mMKH₂PO₄, 50.7 mM Na₂HPO₄, 0.1M NaCl, 0.5% phenol red) for one hour atroom temperature. The detecting layer consisted of polyclonal rabbitanti-human suPAR (1 μg/mL) in dilution buffer. The secondary antibodyused was a monoclonal anti-rabbit immunoglobulin conjugated withalkaline phosphatase (Sigma), diluted 1:2000 in dilution buffer. Thirtyminutes after adding the substrate (1 tablet p-nitrophenyl phosphate,Sigma) in 12 mL substrate solution, pH 9.5 (0.1M Tris base, 0.1M NaCl, 5mM MgCl₂), the reactions were stopped using 50 μL/well 1M NaOH andmeasured at 405 nm. Three laboratory standards were included on allplates. Inter-assay and intra-assay variations were less than 10%. Thedetection level of suPAR was 0.03 ng/ml.

Statistics

All statistical analyses were performed using the statistical programSSPS, Version 10 or SAS. For comparisons between groups, the MannWhitney U test or students T-test were used. The probability of suPAR topredict active TB was assed using a logistic regression model. Allprobability-tests were performed as likelihood ratio tests. Differencebetween Kaplan-Meier curves was analysed by the log-rank test. Theability of serum suPAR to predict mortality in the context of otherknown prognostic markers was formally assessed using the Coxproportional hazards model. All test were conducted as partiallikelihood ratio tests. The statistical model allowed for other thanlinear relationship of suPAR to mortality. The linear relationship wassufficient. A significance level of 5% was used. Cox regression analysiswas carried out from time of inclusion and until treatment terminationor censoring due to death (N=30), beginning of the war in Guinea Bissau(Jun. 6, 1998) (N=50), or loss to follow-up because the patient movedback to the rural areas (N=23). The sixteen patients, who were receivingTB treatment at time of enrolment, were not included in the survivalanalysis since treatment may have affected their suPAR level. RegardingHIV-status, patients dually infected (both HIV-1 and HIV-2 positive)were included in the analyses as HIV-1 positive since dually infectedindividuals have been found to have the same risk and severity of TBinfection as HIV-1 individuals.

Ethics

Pre and post-counselling for both HIV result and TB disease was offeredto all included by the doctors in charge of the study. The subjects wereinformed in writing in Portuguese and verbally in their own languagebefore being enrolled in the study. Informed consent was obtained fromall patients. The study was approved by the Ministry of Public Health inGuinea-Bissau, and by the Central Ethical Committee of Denmark.

Results:

All 262 individuals had measurable suPAR and the median suPAR level was2.1 ng/ml (range 0.66-18.7 ng/ml) at enrolment. There was no correlationbetween age and suPAR (p=0.9) and no difference in suPAR levels betweenmen and women (p=0.87)(table 1). Of the 262 individuals, 16 were alreadyknown to have active TB and were under appropriate treatment at time ofenrolment. Of the remaining 244 individuals, active TB was diagnosed in182, who consequently entered the 8-month treatment regimen. Amongthese, 84 were found positive by direct microscopy of sputum (TB sputumpositive) and 35 cases were negative in direct microscopy but positivein culture (TB culture positive) and 63 were diagnosed on clinical andradiological grounds (presumed TB). The remaining 64 were diagnosed ashaving pneumonia or bronchitis (TB negative).

suPAR is Elevated in Tuberculosis Infection

The suPAR levels were significantly higher among the TB positivepatients (TB sputum positive, TB culture positive and presumed TB,N=182) compared to the TB negative patients (N=64)(p<0.001) (FIG. 1).The suPAR levels were elevated in the 84 TB sputum positive (p<0.001),and in the 35 TB culture positive (p=0.005) compared to the 64 TBnegative. There was a trend towards elevated suPAR levels in the 63diagnosed presumed TB compared to the TB negative (p=0.06). Nodifference was observed in suPAR levels between the 16 patients who hadreceived treatment prior to inclusion in the study and the TB negative(p=0.72, t-test). The suPAR levels are shown in FIG. 1.

suPAR Carries Diagnostic Value for TB

When including all enrolled in the study, there was a significant higherprobability of being diagnosed with TB with increasing suPAR. This isshown as a probability plot in FIG. 2 a. Due to the difficulties ofdiagnosing the sputum negative individuals, the most interestinganalysis was whether suPAR carried diagnostic value in the 162 sputumnegative individuals, i.e., when excluding the 84 TB sputum positive. Wefound that there was an increasing risk of being diagnosed as TB culturepositive or presumed TB with increasing suPAR levels (p=0.001, FIG. 2b). Because HIV-1 infection affects the suPAR level, we made the sameanalysis for the 130 HIV-1 negative individuals (76 HIV-1 negative TBculture positive or presumed TB and 54 HIV-1 negative TB negativepatients) and found the same association between increasing probabilityof being diagnosed as TB positive with increasing suPAR levels (p=0.02,FIG. 2 c).

Higher suPAR Level at Inclusion Among the TB Bacteriological Patientswho Died During Follow-up

Patients were followed from time of inclusion and were treated for an8-month period. Thirty died during the follow-up with a median suPARlevel at inclusion of 3.26 (range 1.04-18.70), which was significantlyhigher than among the 232 survivors (median suPAR 2.12, range0.66-16.22)(p=0.02). Among the 119 bacteriologically confirmed TBpatients (TB sputum and TB culture positive), ten died during the followup. Those who died had significantly higher suPAR levels (median 5.98ng/ml, range 1.3-18.3) than the survivors (median 3.04 ng/ml (Range0.9-14.7)) (Mann Whitney, p=0.022). Among the 63 presumed TB, 13 diedduring the treatment period. The suPAR level at inclusion of the 13 whodied was median 2.35 ng/ml (range 1.0-6.6) versus median 2.12 ng/ml(range 0.7-6.6) among survivors (p=0.45). Among the 64 TB negative, 7died during the follow-up with median suPAR of 3.32 (range 1.43-18.70)and 57 was alive at follow-up having a median suPAR of 1.17 ng/ml (range0.81-16.22)(p=0.04). When dividing the patients into 4 equally sizedgroups based on suPAR level, there was a trend towards highermortalitity among patients in the highest suPAR quartile compared to thelowest suPAR quartile (5/65 vs. 13/65, p=0.07)(table 1).

Kaplan Meier Analysis on Patients with Active TB

There was no difference in survival among patients with active TB (TBsputum, TB culture or presumed TB, n=182) when dividing patients bymedian suPAR value (FIG. 3A). Patients with more than two times themedian value, i.e., patients having higher than 4.2 ng suPAR/ml serum(n=38) were at increased risk of dying during the follow up compared topatients having suPAR values below 4.2 ng/ml (N=144)(MR=3.05 per ngsuPAR increase, p=0.007), FIG. 3B.

suPAR Level as Predictor for Outcome of HIV-1 Infection

47 were found to be HIV-1 or dually infected at time of inclusion. TheHIV-1 infected had a median suPAR level of 2.34 ng/ml (range 0.92-18.7).Of the 47 patients, 13 died during the follow-up (median suPAR 3.43,range 1.15-18.70). These patients had significantly higher suPAR valuethan the 34 patients alive at study censoring (median suPAR 2.05. range0.92-6.88) (p=0.015, Mann Whitney). Cox regression analysis on HIV-1infected showed that suPAR was significantly associated with survival inthis subgroup (p<0.001, MR=1.53). There was a similar difference insuPAR levels among the 66 HIV-2 infected individuals (median suPAR 2.44,range 0.91-18.30), of whom 7 died during follow-up (median suPAR 3.32,range 1.04-18.3), 59 survived (median suPAR 2.44, range 0.91-14.71)though the difference was not significant due to the smaller number ofdeaths (p=0.64). There was no significant difference between suPARlevels in patients with TB with or without HIV infection or between thenumber of HIV 1 or 2 infected between the different TB diagnostic groups(Table 2).

SuPAR Serum Level is Correlated to Survival in Cox Regression Analysis

Excluding the 16 patients who had received treatment at enrolment, 182patients diagnosed with active TB were followed for a period of up to 8months after initiation of treatment and 23 died. In univariate Coxregression analyses, suPAR levels were significantly associated withdeath during treatment (increase in mortality rate ratio (MR)=1.18 perng suPAR increase, p=0.01), as was HIV-1 infection (MR=2.66, p=0.023)and status as TB positive based on clinical and radiological grounds(presumed TB) (p=0.03). When treating all TB positive patients equallyregardless of their HIV-status we found no effect of diagnostic methodon time to death when taking into account the level of suPAR (p=0.43).Neither HIV-2 positivity (p=0.63), age (p=0.72), sex (p=0.19) or log 10transformed CD4 T cell counts (p=0.42) were found to be significantlyassociated with survival during the follow-up period.

In a multivariate Cox analysis including all significant factors, allremained significant the MR being 3.9 (p=0.003) for bacteriologicalnegative TB (presumed TB) compared with the bacteriological positive TBpatients, MR=2.1 (p=0.12) for HIV-1 positivity compared with HIV-1negative TB patients, and 1.28 (p<0.001) per ng suPAR increase.

When excluding the HIV-1 positive patients, 14 died among the remaining149 TB patients. Among these HIV-1 negative patients with active TB,suPAR still retained predictive power (RR=1.14, p=0.05). Thus, weobserved the same dose/response (suPAR/time to death) relationship whenexcluding the HIV-1 positive patients.

Discussion

Mycobacterium Tuberculosis affects the lives of millions of peopleworldwide and 3 millions are estimated to die from the disease everyyear. In this example we find, for the first time, that the suPARmolecule holds diagnostic and prognostic value in TB infection.

In this example, 32 percent of the 262 individuals suspected to be TBpositive were found positive by direct microscopy. These TB sputumpositive had higher levels than those diagnosed by culture (TB culturepositive) or clinical diagnosed (presumed TB). All these patients hadhigher suPAR levels than the TB negative.

Due to the difficulties of diagnosing active TB in patients negative inmicroscopy, an important question in this study was whether suPAR mayhave diagnostic value in individuals in who suspicion of TB remains. Wefound that there was an increasing probability of patients beingdiagnosed as having active TB by culture (culture positive) or byclinical investigation (presumed TB) with increasing suPAR level. The TBnegatives were included in the study on suspicion of TB due to relatedsymptoms and were regarded as having pneumonia after negative tests inculture and clinical examination and treated with antibiotics postentry. We do not know whether the suPAR level may be higher among the TBnegatives compared to a control group of healthy individuals. However,there was a marked difference between the TB negatives and the groupsdiagnosed as having active TB indicating that pneumonia would not causea major increase in suPAR.

Sixteen patients were receiving appropriate TB treatment at time ofinclusion. The serum level of suPAR in these patients was significantlylower than in the patients diagnosed with TB after inclusion, andcomparable to the TB negative. This indicates that the suPAR level maydrop following treatment and that suPAR can be used to monitor theeffect of treatment.

SuPAR was prognostic for survival in this cohort. Among the TB sputumand culture positive, 10 died during the follow-up, and theseindividuals had significantly higher suPAR values at inclusion. UsingCox regression analysis, suPAR was significantly associated withsurvival among the 182 patients diagnosed with TB. We have previouslyshown suPAR to be prognostic for HIV-1 disease progression and thisobservation was confirmed in this study. When excluding the HIV-1 anddually positive, suPAR still remained significantly associated withsurvival among the patients with active TB. Another factor significantlyassociated with survival in Cox regression analysis is type of TBdiagnosis. Patients diagnosed of active TB by clinical examination only(presumed TB) were at larger risk of dying during the follow up whichprobably is due to the delayed treatment. The observation of highestsuPAR level in the TB sputum positive followed by TB culture positiveand TB individuals negative in bacteriological assays (presumed TB)indicated that suPAR might be correlated to the tuberculosis bacterialload. As increased suPAR levels are likely to reflect increase uPARexpression on cell surface Mustjoki our findings suggests that eithersuPAR and/or uPAR may be involved in the pathogenesis of tuberculosisinfection. The initial interaction between M. Tuberculosis and hostmacrophages is an important first step in the pathogenesis oftuberculosis. This step is mediated by specific monocyte/macrophagereceptors and ligands present on the surface of TB. Several macrophagereceptors has been reported to mediate binding to M. Tuberculosis,including complement receptors CR1, CR3 and CR4, glucan receptor,mannose receptor lipopolysacharide receptor and toll-like receptors.However, none of these receptors have been demonstrated to be essentialfor the macrophage/M. tuberculosis interaction. The involvement of CR3in tuberculosis entry have been further enlightened by the use of CD11bknock-out mouse models which show a 60% reduction in tuberculosisinfectivity but no effect on intracellular replication. uPAR have beenshown to complex with CR3 (mac-1, CD11b/CD18). uPAR bind CR3 throughCD11b (reviewed in 14 resulting in activation of the complex. Thus, onepossible reason for the diagnostic and prognostic role of suPAR in TBcould be due to involvement and enhancement of uPAR/CD11b tuberculosiscell entry. Another possibility is that uPAR become upregulated byintracellular TB replication and thereby reflects the number of infectedcells. Apart from HIV and now tuberculosis infection, the serum level ofsuPAR is also a prognostic marker for overall survival in patientssuffering from ovarian and colorectal cancer and for the response totherapy in leukaemia¹⁵,¹¹. Another possible suPAR elevator is borreliaburgdorferi, as a resent in vitro study showed increased levels of uPARon monocytic cells following infection. To be able to compare databetween individual studies in cancer, TB and HIV infection, it isnecessary to develop a reproducible and commercially available suPARassay. The assay used in this study is comparable to the assay used inour previous study of suPAR as prognostic factor in HIV progression⁷ anddescribed in the study by Stephens et al.¹⁶

In conclusion, we find that suPAR is elevated in patients with active TBand that patients with high suPAR level has a higher risk of dyingduring the treatment period.

TABLE 1 Patients divided into four equally sized groups according totheir suPAR level. Brackets show number of patients that died duringfollow-up. First Second Third Forth quartile quartile quartile quartileMedian Median Median median 1.21, 1.84, 2.90, 5.10, range range rangerange 0.66-1.51 1.51-2.18, 2.23-3.43, 3.52-18.70 N = 65 N = 66 N = 66 N= 65 Total N TB  9 (1) 17 (1) 27 (1) 31 (6)  84 (9) sputum positive TBculture  6 (0) 10 (0)  7 (0) 12 (1)  35 (1) positive Presumed 16 (2) 17(4) 18 (4) 12 (3)  63 (13) TB TB 27 (2) 17 (1) 12 (2) 8 (2)  64 (7)negatives Treatment  7 (0)  5 (0)  2 (0)  2 (0)  16 (0) at inclusionTotal 65 (5) 66 (6) 66 (7) 65 (12) 262 (30) HIV-1 10 (2) 11 (0) 12 (5)14 (6)  47 positive HIV-2 14 (1) 16 (2) 18 (1) 18 (3)  66 positive Age44 39 39  36 (median) Men 37 36 38 36 147 Women 28 30 28 29 115

TABLE 2 The table illustrates the number (and percentage of TBdiagnostic group) and suPAR (median, range) of HIV infected amongpatients grouped according to TB diagnosis. Diagnose, N (% of group)suPAR median (range) HIV-1 infected HIV-2 infected HIV negative DeadSputum positive,   11 (13.1%)   24 (28.6%)   49 (58.3%)   9 (10.7%) N =84 3.11 (1.8-10.9) 3.28 (1.3-18.3) 3.09 (1.0-13.4) 5.86 (1.3-18.3) 3.17(0.99-18.30) Culture positive,     (25.7%)   6 (17.1%)   20 (57.1%)   1(2.9%) N = 35 2.23 (1.2-7.0) 3.07 (0.9-6.7) 2.25 (1.1-8.2) 7.01 2.41(0.91-8.21) Presumed TB,   13 (20.6%)   19 (30.1%)   31 (49.2%)   13(20.6%) N = 63 2.50 (1.2-6.8) 2.08 (1.0-6.0) 2.13 (0.7-5.3) 2.35(1.0-6.8) 2.13 (0.66-6.77) Treat at enrol,   4 (25%)   1 (6.3%) 68.8%)  0 N = 16 1.79 (1.4-3.3) 1.53 1.29 (0.8-7.3) 1.62 (0.77-7.30) TBnegatives,   10 (17.9%)   16 (25.2%)   38 (56.9%)   7 (10.9%) N = 641.67 (0.9-18.7) 2.15 (1.0-4.6) 1.63 (0.8-16.2) 3.32 (1.4-18.7) 1.73(0.81-18.70)

Example 2

The serum level of soluble urokinase receptor is elevated inTuberculosis patients, predicts mortality during treatment and may beused to monitor TB treatment efficacy.

Objective:

To investigate whether the serum level of soluble urokinase plasminogenactivator receptor (suPAR) carries prognostic information in individualsinfected with Mycobacterium tuberculosis during the treatment period andwhether it may be used to monitor TB treatment efficacy.

Design:

suPAR was measured by ELISA in 262 individuals at time of enrolment intoa cohort based on suspicion of active tuberculosis and in 101individuals after 8-months of follow-up. The 262 individuals are thesame as investigated in Example 1.

Results:

The suPAR levels were elevated in patients with active TB compared to TBnegative individuals (p<0.001). suPAR levels were highest in patientspositive for TB in direct microscopy (N=84, median suPAR 3.17 ng/ml,p<0.001), followed by patients negative in direct microscopy but culturepositive (N=35, median suPAR 2.41 ng/ml, p=0.005) and by patientsdiagnosed on clinical grounds (N=63, median suPAR 2.13 ng/ml, p=0.06)compared to 64 TB negative individuals (median suPAR 1.73 ng/ml). Duringthe 8-month treatment period, 23 TB cases died. In a multivariate Coxmodel controlling for HIV status, age, sex, CD4 count and type of TBdiagnosis, the mortality increase per ng suPAR was 1.25 (95% Cl1.12-1.40). After treatment, suPAR levels had decreased to the levels ofTB negative individuals.

Conclusions: suPAR levels are elevated in TB patients and associatedwith mortality. Furthermore, suPAR is a marker of treatment efficacy.

Study Population and Methods:

Inclusion Criteria and Laboratory Methods

The subjects included in this study are the same as in example 1. Thedifference between the study described here and the one in example 1, isthat this study investigates the role of suPAR as marker during the 8months of therapy.

Furthermore, during the treatment period, 30 patients died. After 8months of follow-up, a second blood sample was drawn. Of the 262individuals included in this study, 101 individuals were available for asecond bleeding.

Statistics

For comparisons between groups, the Mann-Whitney U-test was used exceptfor comparisons between pre- and post TB treatment suPAR levels in whichcases paired samples t tests were used. The difference betweenKaplan-Meier curves was analysed by the log-rank test. The ability ofserum suPAR to predict mortality, accounting for other known prognosticmarkers, was formally assessed using a Cox proportional hazards modelallowing for non-linear relationships between suPAR and mortality. Alltests were conducted as partial likelihood ratio tests. The analysis oftime to death was carried out from time of inclusion to the first of theevents: treatment termination, 8 months follow-up, death (N=30),beginning of the war in Guinea Bissau which influenced treatment (N=50),or loss to follow-up because the patient moved back to the rural areas(N=23). The sixteen patients who were receiving TB treatment atinclusion were not included in the survival analysis since treatment mayhave affected their suPAR level. Regarding HIV-status, patients duallyinfected (both HIV-1 and HIV-2 positive, N=19) were included in theanalyses as HIV-1 positive since dually infected individuals have beenfound to have the same risk and severity of TB infection as HIV-1individuals. Statistical analyses were performed using the program SSPS,Version 10 or SAS version 8.1. A level of 5% was used for significance.

Ethics

All subjects received written information in Portuguese and verbalinformation given in their local ethnic language, prior to enrolment.Informed consent was obtained from all patients. The study was approvedby the Ministry of Public Health in Guinea-Bissau, and by the CentralEthical Committee of Denmark.

Results:

All 262 individuals had measurable suPAR and the median suPAR level was2.1 ng/ml (range 0.66-18.7 ng/ml) at enrolment. There was no correlationbetween age and suPAR (p=0.9) or in suPAR levels between men and women(p=0.87)(table 1). Of the 262 individuals, 16 were already known to haveactive TB and were under appropriate treatment at time of enrolment. Ofthe remaining 246 individuals, active TB was diagnosed in 182, whoconsequently entered the 8-month treatment regimen. Among these, 84 wereTB sputum positive, 35 cases were TB culture positive, and 63 werediagnosed as presumed TB. The remaining 64 were diagnosed as TBnegative. During the treatment period 30 cases died, of these were 7 TBnegative (Table 1).

suPAR is Elevated in Tuberculosis Infection

The suPAR levels were significantly higher among the TB positivepatients (TB sputum positive, TB culture positive and presumed TB,N=182) compared to the TB negative patients (N=64, p<0.001). The suPARlevel seems to correlate to the number of mycobacterium in sputum as thehighest suPAR levels were found among the 84 TB sputum positive(p<0.001), followed by the TB culture positive (p<0.001), and thepresumed TB (p=0.06) when comparing to the suPAR levels found in the 64TB negatives as shown in FIG. 1. No difference was observed in suPARlevels between the 16 patients receiving TB treatment at time ofenrolment and the TB negative (p=0.72). Grouping of patients intoquartiles according to suPAR level is shown in table 1.

Kaplan Meier Analysis on Patients with Active TB

There was no significant difference in the survival rate among patientswith active TB (TB sputum, TB culture or presumed TB, n=182) whendividing patients by median suPAR value (FIG. 3A). Patients with highsuPAR (more than two times the median value, i.e., patients havinghigher than 4.2 ng suPAR/ml serum (n=38)) died significantly fasterfollowing enrolment, compared to patients below 4.2 ng/ml (N=144,p=0.007), FIG. 3B.

suPAR SERUM Level is Correlated to Survival in Cox Regression Analysis

Excluding the 16 patients who had already started treatment at time ofenrolment, 182 patients diagnosed with active TB were followed for aperiod of up to 8 months after initiation of treatment, 23 of thesedied. In univariate Cox regression analyses, suPAR levels weresignificantly associated with death during treatment, the increase inthe mortality rate ratio (MR) being 1.18 per ng suPAR increase (95% Cl:1.06-1.30), as was HIV-1 infection (MR=2.71, 95% Cl: 1.16-6.29) andstatus as TB positive diagnosed on clinical and radiological grounds(presumed TB) (MR=2.48, 95% Cl: 1.09-5.66). When treating all TBpositive patients equally regardless of their HIV-status and adjustingfor the level of suPAR we found no effect of diagnostic method onmortality (p=0.43). Neither HIV-2 positivity (p=0.63), age (p=0.72), sex(p=0.19) or log 10 transformed CD4 cell counts (p=0.42) were found to besignificantly associated with survival.

All factors found to be significant in univariate analyses remainedsignificant in the multivariate Cox analysis. Controlling for TBdiagnosis (presumed TB compared with TB-cases positive in directmicroscopy or culture, MR=3.5 (95% Cl: 1.39-8.67)), and HIV status(HIV-1 positive compared with HIV-1 negative TB patients, MR=2.5 (95%Cl: 1.07-5.99)), the increase in mortality per ng suPAR was MR=1.25 (95%Cl: 1.12-1.40).

When excluding the HIV-1 positive individuals, 14 died among theremaining 149 TB patients during the follow-up. Among these 149 TBpatients, suPAR still retained predictive power (MR=1.14, 95% Cl:1.00-1.31). Thus, the positive association between suPAR and death wassimilar in HIV-1 positive and HIV-1 negative subjects.

suPAR Level as Predictor for Outcome Among the HIV-1 or -2 InfectedIndividuals

We have previously shown that suPAR is a strong prognostic factor forHIV-1 progression. In the present study, 47 of the 262 individualssuspected of having TB were found to be HIV-1 positive at time ofinclusion and 13 died. Univariate Cox regression analysis on the HIV-1infected showed that suPAR was significantly negatively associated withsurvival (n=47, MR=1.53 per ng suPAR increase, 95% Cl: 1.22-1.92). Noother parameters (age, sex, TB diagnosis or CD4 count) weresignificantly associated with survival in this subgroup. Sixty-six ofthe 262 individuals were HIV-2 positive at inclusion (not including thedually infected) of whom seven died during follow-up. There was asimilar effect of suPAR on survival (n=66, MR=1.13, 95% Cl: 0.97-1.32)although the difference was not significant due to the smaller number ofdeaths. Neither age, sex, TB diagnosis nor CD4 counts were significantlyassociated with survival in the HIV-2 group. There were no significantdifferences in suPAR levels in TB patients with or without HIVinfection, or between HIV-1 and -2 infected individuals in the differentTB diagnostic groups.

Treatment was associated with a decrease in suPAR levels

Of the 262 individuals included in this study, serum samples wereavailable from 101 patients after 8 months of treatment. At inclusion,45 were diagnosed as TB sputum positive, 15 as TB culture positive, 22as presumed TB, 6 received treatment at enrolment, and 13 as TBnegatives. The pre- and post treatment suPAR levels for theseindividuals are shown in FIG. 3. Treatment was associated with asignificant decrease in suPAR among the 45 TB sputum positive (95% Cl:−2.07-−0.56 ng/ml) and a non-significant decrease for the 15 TB culturepositive, (95% Cl: −2.12−0.24 ng/ml). No difference in pre- and posttreatment suPAR values was observed for presumed TB (95% Cl: −0.81-0.84)or for patients receiving treatment at enrolment (95% Cl: −3.3−3.1). Anincrease in suPAR was observed after treatment among the 13 TB negatives(3 HIV-2 positive and 10 HIV negative (p=0.041, 95% Cl: 3.31−1.33ng/ml). The increase was most prominent among the three HIV-2 positive.The post treatment suPAR levels did not differ between the differentdiagnostic groups (FIG. 3).

Discussion

Mycobacterium Tuberculosis affects the lives of millions of peopleworldwide and 2 millions are estimated to die from the disease everyyear. In the present example, we found that suPAR levels were elevatedin TB patients, correlates to type of TB diagnosis and carriesprognostic value. Furthermore, treatment was associated with decreasedserum suPAR levels.

suPAR was found to be significantly higher among the TB cases comparedto the non-TB cases. Furthermore, the highest suPAR levels were found inTB cases positive in direct microscopy, followed by cases negative indirect microscopy but positive in culture, and then the TB casesnegative in both direct microscopy and culture. Given that culture is amore sensitive method than direct microscopy in diagnosing presence ofAFB in sputum, these results may indicate that the level of suPAR inblood is correlated to the number of bacteria in sputum and consequentlythe bacterial load in the bronchi. The enhanced suPAR levels may be aresult of mobilisation of macrophages into the bronchi. Adherence andmigration of monocytes involves a functional interaction between uPARand integrins as also supported by studies in uPAR knock-out mice {May,Kanse, et al. 1998 MAY 1998/id}. The best-described uPAR/integrininteraction is between uPAR and CD11b/CD18 (complement receptor 3 orMAC-1) {Simon, Rao, et al. 1996 14/id}. After this patent was filed,Juffermans and coworkers showed concurrent upregulation of uPAR andCD11b during experimental endotoxemia using LAM, a cell wall componentof M. Tuberculosis ¹⁷. Interestingly, CD11b/CD18 has also been shown tomediate attachment of M. tuberculosis to macrophages¹⁸ suggesting thatTB may exploit this upregulation to adhere and infect recruitedmacrophages. Thus, we propose that this may be a new target for M.tuberculosis interaction.

Using Cox regression analysis, suPAR was found to be significantlyassociated with survival among the 182 patients diagnosed with TB. InGuinea-Bissau, the prevalence of HIV is higher among TB cases comparedto healthy controls. We have previously shown suPAR to be a strongprognostic marker for HIV-1 disease progression¹¹, and this observationwas confirmed in the present study. However, even when excluding theHIV-1 infected, suPAR still remained significantly associated withsurvival among the patients with active TB.

Eight months of treatment lead to a significant decrease in suPARlevels, resulting in similar levels between those diagnosed with orwithout TB at inclusion. Also, sixteen patients were already receivingappropriate TB treatment at the time of inclusion. The serum suPARlevels in these patients were significantly lower than in the patientsdiagnosed with TB after inclusion, and comparable to the TB negative.These observations show that suPAR measurement may be a useful tool formonitoring treatment response in TB patients.

suPAR is elevated and prognostic for outcome in HIV-1 infection. Thisstudy has shown that the serum level of suPAR is elevated in TB patientsand that the pre-treatment level is positively associated with mortalityin patients receiving treatment for TB.

Conclusions:

The serum level of soluble urokinase receptor (suPAR) is elevated inpatients with active tuberculosis and highest in patients who are sputumpositive in microscopic analysis. suPAR was found to positivelyassociated with mortality in TB patients during treatment, even whenexcluding the HIV-1 co-infected for whom suPAR is known to be a verystrong prognostic marker. Following treatment, sputum positive patientshad significantly lower suPAR levels suggesting suPAR could be used tomonitor TB treatment efficacy.

Example 3

The Plasma Level of Soluble Urokinase Plasminogen Activator Receptor isElevated in Patients with Streptococcus pneumoniae Bacteremia andCarries Strong Prognostic Value.

Streptococcus pneumoniae (S. pneumoniae) is the primary agent ofcommunity-acquired pneumonia and to a high degree associated withbacteremia. The annual incidence of pneumonia is estimated from 1 to 12per 1000 population in developed countries. Pneumococcal bacteremiaranges from 9 to 18 per 100,000 per year. Mortality of pneumococcalbacteremia ranges from 17% to 36% with highest mortality in the elderly.

The urokinase-type plasminogen activator receptor (uPAR/CD87) isexpressed on different cell types including neutrophils, lymphocytes,macrophages, endothelial and malignant cells.

It is well known that uPAR is implicated in numerous biologicalfunctions. uPAR and its ligand urokinase-type plasminogen activator(uPA) are involved in converting plasminogen into plasmin. In addition,uPAR binds β integrins as well as signals the recruitment ofinflammatory cells¹⁰. In the pathogenesis of cancer, uPA and uPAR play akey role in tissue invasion by the degradation of extracellular matrix.uPAR can be cleaved from the cell surface by a number of proteases, suchas chymotrypsin, phospholipase C and uPA¹⁹. The proteolytic cleavage ofuPAR from the cell surface releases a chemotactic active form of suPAR.Alternatively, uPAR is released/secreted from the cell surface withoutproteolysis.

Recently, we reported a strong correlation between advancing diseasestate of HIV-1 infection and elevated suPAR suggesting the use of suPARas a prognostic marker. Finally, as shown in this patent application,patients with pulmonary tuberculosis have increased serum levels ofsuPAR.

The possible role of suPAR and uPA in immune responses towards otherinfectious diseases remains unclear. Gene knockout mice lacking uPARhave showed reduced pulmonary neutrophile recruitment and increasedmortality to infection with S. pneumoniae compared to wildtype mice²⁰.Lipotechoic acid from Streptococcus pyogenes facilitates monocyte uPARupregulation in vitro. Upregulation of uPAR by monocytes induced byBorrelia burgdorferi surface proteins has also been reported²¹.

The serum level of the protein YKL-40 has been described elevated duringpneumonia and as an independent prognostic factor in pneumococcalbacteremic patients. Here we investigate whether S. pneumoniaebacteremia affects the plasma suPAR level. We report, for the firsttime, that suPAR is elevated and carries prognostic value in patientswith S. pneumoniae bacteremia.

Subjects and Methods

Patients. Between October 1999 and June 2001, adults (18 years or older)with S. pneumoniae bacteremia admitted to one of five universityhospitals (Aalborg, Aarhus, Odense, Hvidovre or Rigshospitalet) inDenmark were included. Blood samples were drawn at inclusion (hospitaladmission) and clinical data during the time of admission were collectedprospectively, as described (G. Kronborg, N. Weis, H. O. Madsen, S. S.Pedersen, C. Wejse, H. Nielsen, P. Skinhøj, P. Garred, submitted forpublication). Blood was drawn from a group of 30 healthy individuals(laboratory workers) for comparison (control group).

A total of 141 patients, 77 females and 64 males were included. The meanage of the patients was 64 years with a range of 20-99 years. Sixty-sixpercent of the patients had an underlying illness and pneumonia was themost common focus of infection (n=116). Twenty-four patients died inhospital (case-fatality rate =17%). Baseline characteristics of thepatients are given in TABLE 3. All patients received appropriateantibiotic treatment from either admission or when the blood culturebecame positive. The study was approved by the local ethical committees.

ELISA. Maxisorb plates (Nunc, Roskilde, Denmark) were coated andincubated overnight at 4° C. with 100 μl 2 μg/ml murine monoclonalanti-suPAR antibody against human suPAR, diluted in coating buffer (15.1mM Na₂CO₃, 35.7 mM NaHC₃, pH 9.6). Plates were washed with buffer (PBS,0.1% Tween 20) and blocked using SuperBlock (Pierce Chemicals) diluted1:1 in PBS. Plasma samples diluted in dilution buffer (7.3 mM KH₂PO₄,50.7 mM Na₂HPO₄, 0.1 M NaCl, 0.5% phenol red, pH 7.4) were added andplates incubated overnight at 4° C. Bound suPAR was detected using 1001μl 1.0 μg/ml rabbit anti-human suPAR polyclonal ab (a kind gift from dr.Gunilla Hoyer-Hansen) and mouse anti-rabbit polyclonal ab conjugatedwith alkaline phosphatase (Sigma, St. Louis, Mo., USA), diluted 1:2000.Each step preceded by 6 times washing with washing buffer, theantibodies were diluted in dilution buffer and plates incubated 1 h at37° C. Substrate (1 tablet p-nitrophenyl phosphate (Sigma, St. Louis,Mo., USA) in 12 ml 0.1 Tris base, 0.1 M NaCl, 5 mM MgCl₂, pH 9.5) wasadded to the wells and incubated at room temperature for 30 min.Reactions were stopped by adding 50 μl 1 M NaOH per well and absorbancewas read at 405 nm. All ELISA measurements were carried out induplicates. YKL-40 was measured by ELISA.

Statistics. Comparisons between groups were made with Mann Whitney Utest. suPAR as prognostic marker was analysed using logistic regressionanalysis. A p value below 0.05 was considered as significant. Allanalyses were carried out using SPSS software (SPSS, Chicago, Ill.,USA).

Results

suPAR is elevated in pneumococcal bacteremic patients. The plasma levelof suPAR was measurable in all the 141 samples with a median value of5.5 ng/ml (range 2.4-21.0 ng/ml). The median level of suPAR in thecontrol group was 2.6 ng/ml (range 1.54.0 ng/ml). The suPAR levels inthe pneumococcal bacteremic patients were significantly higher than inthe healthy controls (FIG. 5A, p=0.001).

suPAR predicts outcome of the infection. Division of the patients intoone group surviving the infection (n=117) and one group of patientswhere the infection ended with death (n=24) showed significantly highersuPAR levels in the latter group (FIG. 5B; p=0.0001). Of the deadpatients, 79% (n=19) had suPAR levels above the median of 5.5 ng/ml.

The clinical parameters of prognostic importance in terms of death dueto the infection were, as described (G. Kronborg et al., submitted forpublication): cerebral symptoms (confusion, unconsciousness) at time ofadmission (p=0.03), hypotension (p=0.047) and kidney failure (p=0.002);see TABLE 1. The suPAR level was significantly elevated in patients withhypotension (p=0.0001) and renal failure (p=0.0001). suPAR was alsoelevated in the group of chronic alcohol abusers (p=0.0001). Underlyingdiseases were not significantly associated to a deadly outcome ofpneumococcal bacteremia, except for alcoholism (p=0.04).

suPAR is correlated to YKL-40, but not CRP. YKL-40 is a lectin secretedfrom neutrophils and macrophages (25). When comparing suPAR levels withthe YKL-40 levels (n=89), we found a strong correlation between suPARand YKL-40 (Spearman rank correlation coefficient: 0.70, p=0.0001).

Most of the patients had measured their level of CRP. There was nocorrelation between suPAR levels and CRP levels (n=131). High CRP levelswere not prognostic of death.

suPAR is an independent prognostic marker. In a logistic multivariateregression analysis (TABLE 3) including all parameters found to besignificant in univariate analysis (cerebral symptoms, mechanicalventilation, treatment of hypotension, renal failure, alcohol abuse andYKL-40) only suPAR remained significantly associated with death.Patients with plasma suPAR above 10 ng/ml had a mortality rate (MR) of13 (p=0.04) and an increase of 1.31 per ng suPAR (95% Cl: 1.10-1.57),suPAR ranged from 2.4 to 21 ng/ml.

Discussion

In this study, we demonstrate for the first time that suPAR levels arehighly elevated in patients with pneumococcal bacteremia. Elevated suPARlevels have previously been detected in patients with various forms ofmalignant diseases as well as in HIV-1 infection and in related lungdiseases e.g. patients with untreated active tuberculosis.

Plasma suPAR is a strong predictor of death with a mortality rate of1.31 per ng suPAR increase (suPAR varied between 2.4 and 21.0 ng/ml) inpneumococcal bacteremia. From this study we can only draw conclusionsabout the prognostic value of suPAR to patients with pneumococcalbacteremia. Hypotension, kidney failure, cerebral symptoms, chronicalcohol abuse and elevated plasma YKL-40 were all of prognostic value,but only elevated suPAR levels was an independent predictor of death inmultivariate logistic regression analysis. We suggest plasma suPAR as anovel prognostic marker for patients with pneumococcal infections.

It is difficult to compare the suPAR values from the different studiesbecause of inter-assay variations as well as differences in the clinicalconditions of the patients included. However, suPAR seems to be highlyelevated in patients with pneumococcal bacteremia compared to healthycontrols. In a study by Riisbro et al., 53 patients with ovarian cancerhad a median suPAR level of 1.3 ng/ml and healthy subjects had a medianlevel of 0.9 ng/ml giving a rise of 44% in the cancer patients.Pneumococcal patients had a markedly higher increase in median suPAR of112% compared to healthy controls.

We found a close correlation between suPAR and YKL-40. In themultivariate analysis YKL-40 above 500 ng/ml, cerebral symptoms, andhemodialysis were associated with elevated MR, but they were not ofsignificance.

Elevated plasma suPAR has been reported from a group (n=13) of ICUsepsis patients²², leading to speculations that the secretion of suPARis increased during acute inflammation. We compared suPAR levels to CRPand found no correlation. This finding is supported by Slot et al.⁶ whofound no correlation between suPAR and CRP in patients with rheumaticdiseases. Thus, this study find substantial evidence to rule out thepossibility of suPAR being an acute phase reactant.

Evidence of the origin of plasma suPAR remains scarce. However, it isknown that uPAR consists of three domains and proteolytic cleavagebetween domain 1 (D1) and 2+3 (D2D3) renders the chemotactic activedomain D2D3¹⁰. Proteolytic cleavage is mediated by both its own ligand,uPA, and by different proteases, e.g. chymotrypsin. We have used acatching antibody of monoclonal anti-uPAR directed against domain 3 inour ELISA. This means that both intact suPAR and the chemotactic domainD2D3 were detected in our assay. The elevated plasma suPAR seen inpneumococcal bacteremia may be caused by increased secretion fromleukocytes. During pneumococcal pneumonia, cytokines and differentchemotactants are released and increased mortality is associated with ahigh pulmonary interleukin-6 level.

uPAR plays an important role in both innate and acquired immunity. Intransgenic mice lacking uPAR May²³ et al. showed the presence of uPAR tobe crucial for leukocytes to adhere to the endothelium. Knockout micelacking uPAR show a diminished immune response to S. pneumoniae withdecreased neutrophile recruitment to the lung and mice lacking uPA hadan increased immune response to S. pneumoniae ²⁰. These observationssuggest that uPAR is needed for neutrophil recruitment but the mechanismis independent of proteolytic ability. In addition to plasminactivation, uPAR is believed to mediate cell-to-cell interaction throughintegrins and thereby involved in signal transduction. It is welldescribed that invasive bacteria use the host plasmin system to degradeextracellular matrix, e.g. group A streptococci activate plasminogen bystreptokinase. The possible proteolytic role of uPAR/uPA in neutrophiland monocyte migration against Tuberculosis and Streptococcus pneumoniaeneeds still to be investigated.

The reported elevations of plasma suPAR during bacteremia might reflectupregulation of uPAR on neutrophils, monocytes and vascular cells andindicate increased neutrophil and monocyte activity. In conclusion,similar to patients with tuberculosis, plasma suPAR is elevated and anindependent predictor of death in patients with pneumococcal bacteremia.

TABLE 3 Characteristic n % mortality (%) Age, years 18-60 54 39 11 61-7550 35 22 >75 37 26 19 Male 64 45 21 Univariate analysis n MR 95% CI pTreatment of hypotension# 19 3.0 1.0-9.0 0.05 Cerebral symptoms# 41 2.91.1-7.4 0.03 Hemodialysis# 11 8.4  2.3-30.7 0.002 Alcoholism 21 3.01.0-8.5 0.04 YKL-40   0-200 ng/ml 29 1.0 — —  201-500 30 1.5 0.2-9.0 0.6 501- 30 11.8  2.4-58.8 0.003 suPAR   0-5 ng/ml 64 1.0 — —  5.1-10 553.0  0.8-10.1 0.08 10.1- 19 20.6 5.3-80  0.0001 Multivariate analysis nMR 95% CI p Treatment of hypotension# 19 0.1 0-2 0.1 Cerebral symptoms#41 3.7  0.8-17.1 0.09 Hemodialysis# 11 6.4  0.3-118 0.2 Alcoholism 210.8 0.2-6.2 0.8 YKL-40   0-200 ng/ml 29 1.0 — —  201-500 30 0.80.08-7.9  0.8  501- 30 3.9 0.4-35  0.2 suPAR   0-5 ng/ml 64 1.0 — — 5.1-10 55 2.2  0.3-16.8 0.4 10.1- 19 13.0  1.1-158 0.04

Example 4

Correlation Between Smear Positivity for TB and suPAR Level

Aim:

To determine whether suPAR has diagnostic value in TB infection bycomparing to microscopic analysis.

Material and Subjects

69 patients from four suburban areas in Bissau, the capital of GuineaBissau was included in the study. Criterion for inclusion in the studywas one or more of the following symptoms and signs without otherexplanatory disease: persistent cough (>1 month) without improvement onantibiotics, constant or periodic fever for more than 1 month, weightloss, dyspnoea, haemoptysis, nightly sweats or lymphadenopathy. suPARwas measured in sputum from 69 patients. All patients were tested forAcid Fast Bacilli (AFB) in sputum direct microscopy. SuPAR was measuredusing ELISA.

Results

All 69 individuals had measurable suPAR in sputum. Median suPAR was 24.9ng/ml sputum (range 3.1-50.0). Nine patients were positive for AFB indirect microscopy, and these patients had a median suPAR of 50 ng/ml(max in assay, range 9,73-50). This was significantly higher than among60 patients negative for AFP in direct microscopy (median suPAR 20.3ng/ml; range 3.1-50 ng/ml), p=0.002 Mann Whitney test (FIG. 6).

Discussion

This study shows that patients positive for AFB have significantlyhigher suPAR levels than individuals negative for AFB in directmicroscopy. Thus, suPAR carries diagnostic information on TB sputumpositivity in TB patients.

Example 5

Patients with Active TB Have Higher suPAR in Sputum

Aim:

To determine whether suPAR is measurable in sputum and whether patientswho receive therapy has lower suPAR levels than patients who are TBpositive but do not receive therapy.

Materials and Subjects

25 patients from four suburban areas in Bissau, the capital of GuineaBissau was included in the study. Criterion for inclusion in the studywas one or more of the following symptoms and signs without otherexplanatory disease: persistent cough (>1 month) without improvement onantibiotics, constant or periodic fever for more than 1 month, weightloss, dyspnoea, haemoptysis, nightly sweats or lymphadenopathy. suPARwas measured in sputum from 25 patients. All patients had signs,symptoms and x-ray-changes compatible with active TB in the chest andwith findings of Acid Fast Bacilli (AFB) in sputum direct microscopy.Seven of the patients received therapy at the time of sputum samplingand 18 patients received therapy after the sputum sample was taken. Thetreatment consisted of a 4 months intensive phase of daily DirectlyObserved Treatment with Ethambutol, Isoniazid, Rifampicin, andPyrazinamide was followed by a 4 months continuation phase withIsoniazid and Ethambutol collected at the health centre twice per monthby the patient. This treatment regimen was recommended for HIV-infectedindividuals by the national tuberculosis programme in Guinea-Bissau whenthe research project was initiated in 1996. For reasons ofconfidentiality and comparability HIV-infected and uninfectedindividuals received the same treatment. In addition, all patients weregiven Vitamin B complex and Multivitamins daily. Adherence to treatmentwas verified by pill count and an INH urine test at 2, 5 and 8 months offollow-up. Defaulting patients were visited by the nurse and encouragedto continue treatment. Specific HIV drugs or prophylactic treatment forHIV-related diseases were not available in Guinea-Bissau, which is oneof the poorest countries of the world.

Results

18 patients positive for AFB and 7 patients positive for AFB butreceiving treatment. There was significantly higher suPAR levels insputum for the patients whom did not receive therapy compared to the 7TB patients that received therapy (p=0.024) (FIG. 7).

Discussion

In this study, we find that suPAR is measurable in sputum. Mostimportantly, we show that TB patients not receiving therapy hassignificantly higher suPAR levels compared to TB patients after therapyhas been initiated. Thus, initiation of therapy results in a decrease insputum suPAR and sputum suPAR measurement may therefore be used tomonitor TB treatment efficacy.

Example 6

Aim:

To determine whether suPAR is measurable in spinal fluid

Materials and Methods

Spinal fluid was obtained from 23 patients with pneumococcal disease andfrom 1 control. SuPAR was measured using ELISA.

Results.

SuPAR was measurable in spinal fluid from all 24 individuals included inthe study. The median suPAR was 3.49 ng/ml spinal fluid. The controlpatient had a suPAR value of 0.69 ng/ml.

Discussion.

Here we show that suPAR is measurable in spinal fluid. Thus, themeasurement of suPAR in spinal fluid may be used for diagnostic andprognostic purposes.

Example 7

The suPAR level, is not influenced by HCV infection or byAlpha-Interferon and Ribavirin Therapy.

Background.

We have shown that the suPAR (soluble urokinase receptor) levelincreases in patients with HIV, Tuberculosis or s. pneumococcalinfection and that suPAR is an independent highly significant prognosticmarker for these diseases. The aim of this study was to determinewhether suPAR level is altered by HCV infection and treatment. If suPARis modulated by HCV infection, this may influence on the prognosticvalue of suPAR in HIV, Tuberculosis or s. pneumococcal infected patientscoinfected with HCV.

Subjects and Methods:

Forty-seven HCV positive patients were treated with alpha-interferon andribavirin for 12 months. All patients were HIV and HBV negative. Bloodsamples were drawn immediately prior to therapy (T0), and the end oftreatment (T12) and 6-month later (T18). Plasma samples from 30 healthycontrols were included for comparisons. suPAR was measured using ELISAand HCV viral load by RT-PCR.

Results:

There was a significant drop in HCV viral load after therapy (pairedsamples t test, p=0.033). There was no difference in suPAR levelsbetween patients before treatment (Median suPAR: 2.58 ng/ml (range1.15-6.01), after treatment (median suPAR: 2.52 ng/ml (range 1.24-6.26)and at 6-month follow-up (median suPAR 2.43 ng/ml (range 1,21-4,98). Nodifference in suPAR was observed between patients and controls (p=0.68).suPAR was highly correlated at all time points (T0-T12, T0-T18, andT12-T18, all p<0.001, all r>0.67, Pearson correlation). In addition,there was no difference in suPAR between the 30 responders and 19nonresponders (p=0.39).

Conclusion:

The plasma level of suPAR remained stable over time indicating thepossible use of suPAR as a serological marker. The serum suPAR level isnot influenced by HCV infection, HCV titer, by introduction ofalpha-interferon and ribavirin therapy, or by response to therapy. Thesedata suggests that HCV/HIV coinfection does not influence on suPAR as aprognostic marker in HIV infected, Tuberculosis or s. pneumococcalinfected patients individuals.

Example 8

The expression of uPAR on PBMC correlates with serum level of suPAR.

Background.

To determine whether there is a significant relationship between surfaceexpression of uPAR and serum level of suPAR.

Methods:

Blood was collected from 10 healthy blood donors attending the donorclinic at Hvidovre Hospital. The serum was collected and suPARconcentration measured using ELISA. PBMC was collected using Histopaqgradient centrifugation, and the expression of CD87 was measured usingR2 antibody, and a secondary FITC conjugated antibody against mouseantibody using FACS analysis. An idiotypic negative control antibody wasincluded as control of specificity.

Results:

All blood donors had measurable uPAR receptor on the PBMC and measurablesuPAR in serum. The serum suPAR levels significantly correlated with theuPAR surface expression (Pearson correlation, p<0.05) (FIG. 8).

Discussion.

Here we have shown a significant relationship PBMC surface expression ofuPAR (CD87) and the serum level of suPAR. This is in accordance with theobservations of others¹¹ who also find that uPAR on circulating cellscorrelate significantly with the plasma/serum suPAR level. Thus, it istherefore reasonable to assume that the surface expression of uPAR maybe a strong prognostic factor in patients with Streptococcus pneumoniaeor Mycobacterium tuberculosis infection. The CD87 (uPAR) could bemeasured using FACS and thereby add valuable information regardingdiagnosis as well as the patients progression status.

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1. A method of evaluating the progression of the state of a subjectsuffering from pneumococcal bacteremia comprising (a) measuring in vitrothe level of one or more markers selected from the group consisting of(i) urokinase plasminogen activator receptor (uPAR), (ii) solubleurokinase plasminogen activator receptor (suPAR), and (iii) one or moredegradation products of (i) or (ii), in each of a number of biologicalfluid samples from the subject, wherein the samples are obtained atdifferent points in time, (b) comparing the measured levels, wherein adecrease in the levels over time indicates an improvement of the stateof the subject.
 2. A method of evaluating the progression of the stateof a subject suffering from a respiratory bacterial infection comprisinga) measuring in vitro the level of one or more markers selected from thegroup consisting of (i) urokinase plasminogen activator receptor (uPAR),(ii) soluble urokinase plasminogen activator receptor (suPAR), and (iii)one or more degradation products of (i) or (ii), in each of a number ofbiological fluid samples from the subject, wherein the samples areobtained at different points in time, (b) comparing the measured levels,wherein a decrease in the levels over time indicates an improvement ofthe state of the subject.
 3. A method according to claim 2, wherein therespiratory bacterial infection is a Streptococcus pneumoniae infection.4. A method according to claim 2, wherein the respiratory bacterialinfection is a Mycobacterium tuberculosis infection.
 5. A methodaccording to claim 2, wherein the subject is undergoing a treatmentregimen.
 6. A method according to claim 1, wherein the biological fluidsample is a urine sample.
 7. A method according to claim 1, wherein thebiological fluid sample is a serum or blood sample.
 8. A methodaccording to claim 1, wherein the measuring is done using a stick.
 9. Amethod according to claim 1, wherein the measuring is done using ELISA.10. The method of claim 1, wherein the marker is suPAR.
 11. A methodaccording to claim 2, wherein the biological fluid sample is a urinesample.
 12. A method according to claim 2, wherein the biological fluidsample is a serum or blood sample.
 13. A method according to claim 2,wherein the measuring is done using a stick.
 14. A method according toclaim 2, wherein the measuring is done using ELISA.
 15. The method ofclaim 2, wherein the marker is suPAR.
 16. The method of claim 1, furthercomprising the step of simultaneously confirming in vitro for infectionby a pneumococcal bacteria, wherein a decrease in the levels over timein infected individuals indicates an improvement of the state of thesubject.
 17. The method of claim 2, further comprising the step ofsimultaneously confirming in vitro for infection by a respiratorybacterial pathogen, wherein a decrease in the levels over time ininfected individuals indicates an improvement of the state of thesubject.